Medium, component thereof and process employing same for the production of microbial insecticides, such as Bacillus popilliae spores and the like, for the control of Japanese beetles and other insects

ABSTRACT

Char waste water, a waste product in sugar refining, derived by water washing spent animal charcoal (char), is useful as a sporulation medium for the production of Bacillus popilliae spores. Bacillus popilliae spores are useful as a microbial insecticide in the control of Japanese beetles since B. popilliae spores cause milky disease in the Japanese beetle and other insects, such as the European chafer.

This invention is related to microbial pesticides or insecticides. Inone embodiment, this invention is directed to a component of a mediumuseful for the sporulation of the vegetative cells of certainmicroorganisms, such as Bacillus popilliae. In another embodiment, thisinvention is directed to the preparation of a medium useful for thesporulation of the vegetative cells of B. popilliae and likemicroorganisms. In still another embodiment, this invention is directedto a method for the production of spores of B. popilliae and similarbacterial spores.

Microbial or bacterial pesticides or insecticides are known and areproduced and employed commercially in the United States. One of the bestknown bacterial insecticides is based on the bacterium Bacillusthuringiensis. This microorganism is the causative agent of fataldisease in many lepidopterous (caterpillar) insects, e.g., cabbagelooper, alfalfa caterpillar, the so-called imported cabbage worm, tentcaterpillar and gypsy moth. This microorganism, B. thuringiensis, iseasily cultivated in various media and spores and the spores thereof canbe mass-produced by conventional fermentation techniques.

Another microbial insecticide is based on the microorganism B. popilliaewhich causes milky disease of the Japanese beetle and the Europeanchafer. In the production of this microbial insecticide, the spores ofB. popilliae, living insect larva, such as the grub of the Japanesebeetle, are injected with the pathogen, i.e., the spores of B.popilliae. After a short incubation period, the injected larvaecontaining the spores of B. popilliae are ground and mixed with anextending material, such as talc.

The above-described process for the production of the spores of B.popilliae is a difficult, costly process, for more details see U.S. Pat.No. 2,293,890. Various techniques have been disclosed for improving theproduction, collection and utilization of the spores of B. popilliae asa microbial insecticide, see U.S. Pat. Nos. 3,308,038, 3,503,851 and3,616,250. The disclosures of the above-identified patents are hereinincorporated and made part of this disclosure.

It is an object of the invention to provide a component useful in themake-up of a substrate or medium for the sporulation of vegetative cellsof the microorganism B. popilliae.

It is another object of this invention to provide a medium useful foreffecting sporulation of vegetative cells of B. popilliae.

It is another object of this invention to provide a process foreffecting the sporulation of microorganisms, particularly bacteria,which undergo sporulation.

How these and other objects of this invention are achieved will becomeapparent in the light of the accompanying disclosure. In at least oneembodiment of the practices of this invention, at least one of theforegoing objects will be achieved.

It has been discovered that char waste water is useful as a component ofa medium for the sporulation of spore-producing microorganisms, such asB. popilliae.

Char waste water is a waste product of sugar refining, such as canesugar refining. In the refining of cane sugar, animal charcoal (char),such as bone char, which is the granular residue obtained by thedestructive distillation, is employed. In the processing of sugarliquors or syrups, after clarification by defecation, the remainingcolor bodies and other impurities in the sugar syrups or liquors must beremoved before a satisfactory refined crystallized sugar is produced.These color bodies and other impurities are removed from the defecated,filtered sugar syrups and liquors by percolation and filtration of thesugar syrups and liquors through cisterns or tanks filled with animalcharcoal (char). After the ability of the char to remove color and otherimpurities from the sugar syrups and liquors undergoing treatment bycontact with the char, the char cisterns or filters are "sweetened off"or washed with hot water. After about 12 to 14 hours washing, the sugarcontent of the resulting wash water is no longer sufficient to warrantrecovery of the sweet or sugar-containing wash water for the eventualrecovery of sugar therefrom. At that time, the wash water is usuallyturned to waste, the washing of the char to waste continuing for about12-24 hours, more or less, usually about 14-18 hours. During this periodof washing the char to waste, there is produced the char waste wateruseful in the practices of this invention. Analyses of typical charwaste waters and ranges for the components thereof are set forth inaccompanying Table I.

                                      TABLE I                                     __________________________________________________________________________    Analyses of Char Waste Water                                                            Range  Char Waste Water                                                                        Char Waste Water                                   Component % By Wt.                                                                             A -- % By Wt.                                                                           B -- % By Wt.                                      __________________________________________________________________________    Sucrose   0.3-2.5   0.60%     1.58%                                           Reducing Sugars                                                                         0.03-0.15 0.054%    0.082%                                          Ash Sulfated                                                                            0.3-0.8   0.40%     0.45%                                           Ash Thermal                                                                             0.15-0.4  0.255%    0.256%                                          Solids (Drying)                                                                         1.1-2.1   1.30%     1.58%                                           ppm Calcium                                                                              500-1500 792       850                                             ppm Magnesium                                                                            60-150   93.8      105                                             ppm Sodium                                                                              20-90     32.5       64                                             ppm Potassium                                                                            50-500   117       247                                             ppm Iron  0.2-9.0   1.3       3.92                                            ppm Copper                                                                              0.4-0.9   0.61      0.57                                            ppm Zinc  0.05-0.3  0.18      0.12                                            ppm Manganese                                                                           0.1-0.8   0.33      0.29                                            ppm Sulfates                                                                             100-1200 282       689                                             ppm Phosphates                                                                           6-40     15.96      13                                             ppm Chlorides                                                                            1-60     2.1       13.2                                            __________________________________________________________________________

As indicated hereinabove, bone char is employed in sugar refineryoperations to remove color bodies and other impurities from sugar syrupsjust prior to crystallization of sugar therefrom. Bone char is usuallymade by heating degreased, crushed and grated animal bones in a closeretort (in the absence of air) to a cherry red heat until most of theorganic matter has been destroyed, volatilized or driven off. Thestructure of the bone char consists of a porous framework of calciumphosphate coated with carbon and is composed structurally of calciumphosphate and some calcium carbonate. The carbon content is made up ofsubstantially elemental carbon and chemically combined carbon. A percentby weight chemical analysis of bone char is substantially as follows:carbon 8.5-10, insolubilized ash 0.2, sulfate (as CaSO₄) 0.08, sulfide(as CaS) 0.07, carbonate (as CaCO₃) 7.9, iron 0.07, phosphate (as CaPO₄)80.6-84.1.

In the washing to spent bone char after the sweetening off operationwhich is primarily carried out to displace the sugar-containing liquorfrom the char cisterns or filters, the bone char having completed itsprimary function of absorbing selected impurities into its high porousstructure, and when the amount of dissolved sugar in the sweet-waterwashing reaches a low level or concentration which does not justify therecovery of sugar therefrom, the washing operation is continued for anextended period of time up to about 24 hours, more or less, withrelatively hot water, such as water having a temperature in the rangefrom about 130°-200°F., to remove the adsorbed impurities, primarily ash(salts) and loosely absorbed color bodies. Upon completion of the waterwashing operation with the production of char waste water, the resultingwashed bone char is then burned in the kiln to remove any residualimpurities and to regenerate. the bone char.

The analyses of typical char waste waters of Table I show a very lowlevel of carbohydrates (primarily sucrose and invert sugar). Theanalyses, however, show a wide range of inorganic ions which areindispensible for the sporulation of various Bacillus species. It isknown that inorganic ions are essential for the formation of bacterialspores and the level or concentration of the inorganic ions required forsporulation varies with the microorganism, the concentration ofnutrients, oxygen supply, temperature and pH. The specific function ofminerals or inorganics in sporulation, however, is unknown. It has beenspeculated that inorganic ions act as catalysts in the activation of themany enzyme systems involved in sporulation.

Of the metals or inorganic ions, manganese has been shown to be requiredfor sporulation, see the article by J. Charney et al. entitled"Manganese as an Essential Element for Sporulation in the GenusBacillus," appearing in J. Bact. 62, 145-148 (1951). Calcium also isrequired for the formation of resistant spores and is necessary at thetime of sporangium formation. Calcium is also known to be required forthermoresistance and in this capacity is closely associated withdipicolinic (DPA), see the article by H. O. Halvorson et al. entitled"The Role of DPA in Bacterial Spores II," 149-164 (1961) published byBurgess Publishing Co., Minneapolis, Minn. Copper has been associatedwith various enzymatic reactions as a co-factor or as a metal ionactivator and the need for iron and zinc for sporulation has beenreported, see the article by H. R. Curran et al. entitled "The Influenceof Iron or Manganese Upon the Formation of Spores by Mesophilic Aerobesin Fluid Organic Media," appearing in J. Bact. 67, 489-497 (1954). Ithas also been reported producing a medium deficiency in potassium,phosphates and comprises prevents most Bacillus from sporulating orreduces the number of spores reduced, see the article by J. W. Foster etal. entitled "Mineral Deficiencies in Complex Organic Media as LimitingFactors in the Sporulation of Aerobic Bacilli," appearing in J. Bact.57, 613-615 (1949).

The relationship of charcoal and increased sporulation was noted by J.L. Roberts et al in the article entitled "Spore Formation by Bacillussubtilis in Peptone Solution Altered by Treatment with ActivatedCharcoal," appearing in J. Bact. 44, 653-659 (1942); also, the articleby J. W. Foster et al entitled "Antisporulation Factors in ComplexOrganic Media. I. Growth and Sporulation on Bacillus larvae," appearingin J. Bact. 59, 463-470 (1950), reported an increase in the sporulationpercent of B. larvae by treating several bacteriological media withactivated charcoal. These investigators noted that charcoal treatmentwas conceivably more effective in enhancing sporulation than it was inincreasing vegetative growth. K. H. Steinkrous et al. in their paperentitled "Studies on the Milky Disease Organisms," Journal Paper No.1086, New York State Agricultural Experimental Station, Geneva, N.Y.,reported that when activated charcoal was added to a solid triptone agarmedium, spores of B. popilliae could be produced and W. C. Haynes et al.reported in their article entitled "Spore Formation by Bacilluspopilliae in Liquid Medium Containing Activated Carbon," appearing in J.Bact. 91, 2270-2274 (1966), the successful sporulation of B. popilliaein a liquid medium containing activated carbon; however, low sporeyields, about 3%, were reported. Also, W. C. Haynes et al. in theirarticle entitled "Hydrolyzed Casein Enhances the Sporogenicity of YeastProducts for Bacillus popilliae in Liquid Cultures," Abstracts of AnnualMeeting, G145 American Society for Microbiology (1973), reported that B.popilliae NRRLB 2309S formed as many as 3.1 × 10⁷ refractile ofresistant spores/ml in a liquid shaken culture containing glucose, K₂HPO₄, activated carbon, yeast and casein hydrolysate. In none of theabove-identified publications, the disclosures of which are hereinincorporated and made part of this disclosure, is there any teaching orsuggestion of the utility of char waste water, such as described aboveand/or produced in a cane sugar refining operation, as a component of asporulation medium, particularly for the sporulation of B. popilliae.

The following is exemplary of the practices of this invention. Bacilluspopilliae NRRLB-2309, in the form of a 4% inoculant of 72 hour frozenspore cultures, was inoculated into three 25 ml portions of a medium forthe vegetative growth of B. popilliae. The medium for vegetative growthcomprised 1.5% yeast extract, 0.3% K₂ HPO₄, 0.2% glucose and 0.5%triptose, the pH of the medium having been adjusted to 7.2-7.3 andsterilized for 15 minutes at 120°C. The inoculated flasks were incubatedon a rotary shaker at 200 rev/min at 27°C. for 24 hours. The resultingcultured vegetative cells were centrifuged and washed three times toremove all traces of the vegetative growth medium. 6.5 ± 0.5 grams ofthe washed vegetative cells of B. popilliae were then employed toinoculate 5 liters of sterile filtered char waste water, the char wastewater having first been filtered through glass wool, the pH adjusted to7.0 and sterilized for 40 minutes at 121°C. The fermentation of theinoculated char waste water was carried out in a 14 liter New BrunswickScientific Co. Micro Ferm laboratory fermentor. The controls of thefermentator were set for the introduction of 0.2 vol per vol per minuteof sterile air, the agitator set for 200 rpm and the temperaturemaintained at 25°C. with no pH control. The inoculated char waste waterwas incubated for a period of 72 hours.

Viable (vegetative) and spore counts were made at 24 hours intervals andthe spores were heated at 75°C. for 15 minutes. The above-describedvegetative growth medium solidified with 2% agar was used for bothviable and spore counts by the membrane filtration method. Microscopicstains were made to determine spore characteristics and DPAdeterminations were made of the harvested spores. The spores wereharvested by the addition of 50 grams of talc, U.S.P., to the 5 litersof the inoculated char waste water after the 72 hour inoculation period.The incubated talc-containing char waste water was then centrifuged andthe talc-spore mixture recovered and dried for 24 hours at 45°C. atsubatmospheric pressure.

In the practice of this invention, the char waste water need not be acomposite of the total char waste water. It has been found that the charwaste water collected at various times during the water washingoperation producing the resulting char waste water produced char wastewater satisfactory for use in a sporulation medium in accordance withthis invention. Indeed, the char waste water samples collected duringportions of the washing operation or samples of the total char wastewater all yielded 100 percent sporulation.

The two-stage fermentation procedure described hereinabove employingcane sugar refinery char waste water as a sporulation medium greatlyexceeded the sporulation rates of the various reported charcoal media asindicated in accompanying Table II.

                  TABLE II                                                        ______________________________________                                        Reported Sporulation Rates of                                                 Charcoal-Containing Media                                                     Charcoal Media       Percent Sporulation                                      ______________________________________                                        J Medium -- 10% Charcoal *                                                                         18.8%                                                    J Medium -- 10% Charcoal *                                                                         8.6%                                                     MYPT Medium -- 10% Charcoal **                                                                     0.13%                                                    S-P Medium -- 3% Charcoal ***                                                                      7.5%                                                     S-P Medium -- 3% Charcoal ***                                                                      13.6%                                                    ______________________________________                                        *    Sporulation of Bacillus popilliae in liquid medium as                         affected by kind of carbon and method of sterilization.                       Haynes, W. C., Weih, L. J., Crowell, C.                                       Can. J. Microbiol. 18:515-518 (1971)                                     **   Characteristics of a new strain of Bacillus popilliae                         sporogenic in vitro.                                                          Sharpe, E. S., St. Julian, G., Crowell, L.                                    Applied Microbiology 681-688 (1970)                                      ***  Studies on the Milky Disease Organisms.                                       Steinkrous, K. H., Provvidenti, M. J. - Journal Paper No. 1086                New York State Agricultural Exp. Station                                      Geneva, New York                                                     

Further, the practices of this invention provide an effective means forproducing large numbers of B. popilliae spores without using complexmedia, prolonged incubation periods and/or other techniques not readilysuitable for large-scale fermentation production.

Although the practices of this invention have been indicated to beapplicable to the production of spores of Bacillus popilliae, thisinvention is generally applicable to the production of microorganisms,particularly bacterial spores. Other spores which are suitably producedin accordance with the practices of this invention include the spores ofBacillus lentimorbus which also produce the milky disease in larvalstages of insects. Those bacteria which form spores, particularlyendospores, are usefully employed in the practices of this invention.Those bacteria which form endospores appear to be limited to a smallgroup of eubacteria family Bacillaceae, and Vibrio spirilla andsarcinae, see the publication by S. T. Lyles entitled "Biology ofMicroorganisms," pages 195-196, published 1969 by The C. V. Mosby Co.,St. Louis, Mo. The above-identified pages of this publication are hereinincorporated and made part of this disclosure.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many modifications, alterations and substitutionsare possible in the practice of this invention without departing fromthe spirit or scope thereof.

We claim:
 1. A method of sporulating a microorganism capable ofundergoing sporulation which comprises incubating said microorganism ina medium containing filtered cane sugar refinery animal char wastewater.
 2. A method in accordance with claim 1 wherein said microorganismis Bacillus popilliae.
 3. A method in accordance with claim 1 whereinsaid microorganism is Bacillus lentimorbus.
 4. A method in accordancewith claim 1 wherein said microorganism is Bacillus popilliae andwherein said microorganism is incubated in said medium at a temperatureof about 27°C. for a period of about 24 hours.
 5. A method in accordancewith claim 4 wherein the resulting produced spores are recovered fromthe incubation medium by the addition of finely divided talc to theincubation medium and a resulting solids admixture consistingessentially of talc and spores separated and recovered from saidincubation medium.
 6. A method of producin spores of Bacillus popilliaewhich comprise effecting vegetative growth of Bacillus popilliae in agrowth medium comprising 1.5% by weight yeast extract, 0.3% weight K₂HPO₄, 0.2% by weight glucose and 0.5% by weight triptose, the pH of saidgrowth medium having been adjusted to a value in the range about7.2-7.3, said growth medium having been sterilized before being employedfor the vegetative growth of Bacillus popilliae therein, recovering theresulting produced cells of Bacillus popilliae, introducing therecovered cells of Bacillus popilliae into an incubating medium foreffecting sporulation of said cells, said incubating medium comprisingfiltered cane sugar refinery animal char waste water, incubating thecells in said incubating medium to induce sporulation of said cells andrecovering the resulting Bacillus popilliae spores.
 7. A method inaccordance with claim 6 wherein said cane sugar refinery waste water hasthe composition:

    Component         Range % By Wt.                                              ______________________________________                                        Sucrose           0.3-2.5                                                     Reducing Sugars   0.03-0.15                                                   Ash Sulfated      0.3-0.8                                                     Ash Thermal       0.15-0.4                                                    Solids (Drying)   1.1-2.1                                                     ppm Calcium        500-1500                                                   ppm Magnesium      60-150                                                     ppm Sodium        20-90                                                       ppm Potassium      50-500                                                     ppm Iron          0.2-9.0                                                     ppm Copper        0.4-0.9                                                     ppm Zinc          0.05-0.3                                                    ppm Manganese     0.1-0.8                                                     ppm Sulfates       100-1200                                                   ppm Phosphates     6-40                                                       ppm Chlorides      1-60.                                                      ______________________________________                                    


8. A method in accordance with claim 6 wherein said cane sugar refinerywaste water has the composition:

                          Char Waste Water A                                      Composition             % By Wt.                                              ______________________________________                                        Sucrose                 0.60%                                                 Reducing Sugars         0.054%                                                Ash Sulfated            0.40%                                                 Ash Thermal             0.255%                                                Solids (Drying)         1.30%                                                 ppm Calcium             792                                                   ppm Magnesium           93.8                                                  ppm Sodium              32.5                                                  ppm Potassium           117                                                   ppm Iron                1.3                                                   ppm Copper              0.61                                                  ppm Zinc                0.18                                                  ppm Manganese           0.33                                                  ppm Sulfates            282                                                   ppm Phosphates          15.96                                                 ppm Chlorides           2.1.                                                  ______________________________________                                    


9. A method in accordance with claim 6 wherein said cane sugar refinerywaste water has the composition:

                          Char Waste Water B                                      Component               % By Wt.                                              ______________________________________                                        Sucrose                 1.58%                                                 Reducing Sugars         0.082%                                                Ash Sulfated            0.45%                                                 Ash Thermal             0.256%                                                Solids (Drying)         1.58%                                                 ppm Calcium             850                                                   ppm Magnesium           105                                                   ppm Sodium              64                                                    ppm Potassium           247                                                   ppm Iron                3.92                                                  ppm Copper              0.57                                                  ppm Zinc                0.12                                                  ppm Manganese           0.29                                                  ppm Sulfates            689                                                   ppm Phosphates          13                                                    ppm Chlorides           13.2.                                                 ______________________________________                                    


10. A method in accordance with claim 6 wherein said spores arerecovered by the addition of finely divided insoluble, inert solidsmaterial to said incubating medium and separating therefrom theresulting solids admixture containing said solids material and saidspores.